Genetic analysis of membrane differentiation in Paramecium. Freeze-fracture study of the trichocyst cycle in wild-type and mutant strains.

Abstract

Using a series of mutants of Paramecium tetraurelia, we demonstrate, for the first time, changes in the internal structure of the cell membrane, as revealed by freezefracture, that correspond to specific single gene mutations. On the plasma membrane of Paramecium circular arrays of particles mark the sites of attachment of the tips of the intracellular secretory organelles--trichocysts. In wild-type paramecia, where attached trichocysts can be expelled by exocytosis under various stimuli, the plasma membrane array is composed of a double outer ring of particles (300 nm in diameter) and inside the ring a central rosette (fusion rosette) of particles (75 nm in diameter). Mutant nd9, characterized by a thermosensitive ability to discharge trichocysts, shows the same organization in cells grown at the permissive temperature (18~ while in cells grown at the nonpermissive temperature (27~ the rosette is missing. In mutant tam 8, characterized by normal but unattached trichocysts, and in mutant tl, completely devoid of trichocysts, no rosette is formed and the outer rings always show a modified configuration called "parentheses," also found in wild-type and in nd9 (18~ cells. From this comparison between wild type and mutants, we conclude: (a) that the formation of parentheses is a primary differentiation of the plasma membrane, independent of the presence of trichocysts, while the secondary transformation of parentheses into circular arrays and the formation of the rosette are triggered by interaction between trichocysts and plasma membranes; and (b) that the formation of the rosette is a prerequisite for trichocyst exocytosis.All freeze-fractured natural membranes studied so far display a smooth background that probably represents regions of lipid bilayer (5, 7), on which "particles" corresponding to proteins or lipoprotein aggregates (9, 20) are usually found. These particles generally appear randomly distributed but various types of organized particle arrays have been described: for instance, the arrays involved in 126

Caffeine-Induced Ca 2+ Transients and Exocytosis in Paramecium Cells. A Correlated Ca 2+ Imaging and Quenched-Flow/Freeze-Fracture Analysis

“…We used wildtype (7S) cells, eventually in axenic cultures for the isolation of trichocysts (see below), nondischarge strain nd9 grown at a nonpermissive temperature of 28°C (Beisson et al, 1976), trichocyst-free strain ''trichless,'' tl (Pollack, 1974), and strains tam38 and tam6, with smaller or larger numbers of always nondocked trichocysts floating free in the cytoplasm (Pouphile et al, 1986).…”

“…8 to 10). Such cells maintain their inability to perform exocytotic membrane fusion for several hours, also during analysis at ambient temperature, although their trichocyst contents are capable of decondensation (Beisson et al, 1976;Pouphile et al, 1986 tion (see Introduction and below). Without Ca o 2+ added, the cortical increase is variable and no central [Ca 2+ ] i rise is recognized (Figs.…”

“…With this method the entire cell surface is stimulated by rapid mixing with caffeine. Opposite to the other strains used, only exocytosis-competent strains like 7S display rosette IMPs in freeze-fracture replicas (Beisson et al, 1976;Pouphile et al, 1986). This well defined morphology was exploited in these studies.…”

Veratridine-Mediated Ca 2+ Dynamics and Exocytosis in Paramecium Cells

“…Wild-type cells (strain 7S), a trichocyst-free strain "trichless" (tl) [39], and a "pawn" mutant (d4-500r) devoid of ciliary voltage dependent Ca channels [20,44] but with normal secretory capacity were all grown at 25°C, the nondischarge mutant nd9 [3,4,30,40] To chelate [Ca 2+ ] o to ∼30 nM, i.e., slightly below resting levels (50-100 nM), we added 4.5 mM EGTA to the extracellular medium, as in ref. [26].…”

“…This normally occurs by influx of Ca o 2+ after formation of an exocytotic pore [37,38]. The nd9 mutant we used was grown at a nonpermissive temperature of 28°C, so it cannot secrete any of its trichocysts, although they are docked in great numbers at the cell membrane [3,4,30,40]. Their trichocyst contents can decondense in vitro, but since nd9-28°C cells cannot form an exocytotic fusion pore, Ca 2+ must artifically obtain access to trichocyst contents to provoke their ("internal") decondensation [40].…”